Determining effects of external stimuli on the brain using pet

ABSTRACT

A method of evaluating the effects of administering an external stimuli or treatment such as a psychoactive compound, a drug, or an environmental influence like temperature, noise, vibration, light and similar sensory-perceived influences, on a subject&#39;s brain using imaging techniques with position emission tomography (PET). The method measures cerebral metabolism before and after administering the external stimuli or treatment, and employs a behavioral clamp to control behavioral influences on the subject&#39;s brain after administration of the external stimuli or treatment.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of application Ser.No. 08/522,685 filed Sep. 1, 1995, now U.S. Pat. No. 5,827,499.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to a method of evaluating theeffects of external stimuli, such as pharmaceutical drugs andenvironmental influences like fragrances, temperature, noise, light,etc. on a subject's brain, and more particularly to a method ofevaluating the effects of administering such stimuli on a subject'sbrain using imaging techniques with position emission tomography (PET).

[0003] Position emission tomography (PET) is a radiotracer based methodfor producing images that quantitatively represent some biochemicalproperty of the body (or portions of the body). In relation to thiswork, use of PET is confined to metabolic imaging of the brain. Althoughother methods are often used, the aspect of PET that is relevant to thisparticular work involves 2-fluoro-deoxyglucose (FDG) as the tracer instudies of cerebral metabolism and oxygen-15 labeled water (0-15) as thetracer in studies of cerebral blood flow. In general, FDG is used toestimate the rate of metabolism of glucose in different parts of thebrain (Sokoloff, 1985) and provides data that represent integratedmetabolic activity over a 20-40 minute period. 0-15 studies determinerate of blood flow in different parts of the brain with an integrationperiod of 40-60 seconds. Given the different temporal demands of the twokinds of tracers, metabolic studies with FDG reveal relativelylong-lasting effects or conditions (such as pathologies), whereas 0-15studies are more sensitive to rapid, transient activity (such as sensoryprocesses or cognition).

[0004] The problem that we are interested in is to determine how certainexternal stimuli or treatments affect cerebral metabolism. The externalstimuli or treatments are usually drugs, but other interventions wouldbe faced with the same considerations. For example, environmentalinfluences such as fragrances, temperature, noise, taste, vibration,light and similar stimuli clearly effect cerebral metabolism. The basicparadigm which we use to study external stimuli or treatments isconceptually very simple: 1) measure metabolism without any externalstimuli or treatment; 2) apply the external stimuli or treatment; 3)measure metabolism again; and 4) determine whether the measurement atstep 3 is statistically different from the measurement at step 1. Inactuality, there are a number of experimental difficulties that must bedealt with before this paradigm can be applied.

[0005] First of all, it is important to realize that all images providedby PET reflect every influence on the brain at the time of a study. Allperceptions, movements, thoughts, and moods, as well as vegetativefunctions, have correlates in brain metabolism and blood flow, and thesefactors, which are always present, may obscure effects due to externalstimuli or treatment. Even more critically, these factors may change inunknown ways in response to the external stimuli or treatment and hencethe extent of their influence on observed metabolism becomesunpredictable. It can, therefore, be difficult to determine whichfeatures of an image are due specifically to the experimental treatmentand which are secondary, due to some other change that occurs because ofthe treatment. The objective of much of the present work has been todevelop ways of processing PET images to more easily identify metaboliceffects that are due to a specific external stimuli or treatment.

[0006] Some of our earliest work involved the recognition that thecondition of subjects at the time of a study might vary from subject tosubject or even within the same subject at different times (Levy et al.,1987). Variation in the testing condition thus could make it difficultto isolate differences introduced by an external stimuli or treatment.

[0007] Accordingly, we have developed an appropriate standard conditionfor testing subjects. This condition is the visual monitoring task(VMT). The VMT requires that subjects watch a screen on which isprojected either a bright light or a dim light. The lights are easilydistinguished from each other. One light flashes at a varying intervalof 4 to 7 seconds. The two lights are equally probable. We ordinarilytest subjects for 3 to 4 blocks (96 total trials each block, about 10minutes per block) with a slight break between blocks. Subjects areinstructed to press a button every time the dim light flashes and toignore the bright flashes (a very subtle point: the natural tendency isto respond to the bright light which is more salient; by making the dimlight the target a slight increase in difficulty is introduced). Acomputer measures reaction time (RT) to each button press (expressed asmedian RT per block) and whether the press was correct (a dim light),false alarm (a bright light), or missing (dim light flashed but subjectdid not press the button). In some situations, the VMT includes afeedback system so that subjects could see how fast their RT's to targetflashes were. This produces more consistent RT's (lower variance). TheVMT differs from other tasks that are occasionally used in PET studies(Buchsbaum et al., 1992; Hazlett et al., 1993) in that it is extremelysimple and undemanding—subjects can do this task even if they are veryyoung, very old, or slightly affected by a drug. At the same time,successful performance of the task precludes extraneous mental activity.

[0008] In early drug/PET work, a common procedure was to use a fairlylarge dose of a drug in order to produce the largest practical metabolicor blood flow “signal”. We, however, immediately recognized that thiswould create a problem. Some of the drugs that we were planning to study(e.g., ethanol, diazepam) would likely incapacitate subjects to thepoint where they would not be able to perform the VMT adequately.However, we were convinced that any dramatic change in behavior as aresult of taking a drug would be impossible to interpret (as an extremeexample, subjects who are sleeping after a drink of ethanol should notbe compared to waking subjects—there would undoubtedly be differences,but these would not be due to the drug but the condition of thesubjects). Therefore, use of the VMT as part of our drug studiesnecessarily limits the dose of some drugs that can be studied. Thus, wechose to sacrifice a large but confounded signal in order to get a smallbut clean signal.

[0009] We also recognized that some subjects became very competitivewhile performing the VMT, visibly trying to get the lowest possible RT.We thus recognized that the demands of the VMT affected differentsubjects differently and perhaps would affect them differently undervarious drug conditions and/or other external stimuli treatments.Therefore, (and as now used in the OMEI process) we eliminated the RTfeedback. Instead, we explicitly adopted an exclusion criterion: anycondition on which RT is not stable (operationally defined as deviatingby more than 10% from a reference condition) must be discardedSimilarly, any subject who does not perform with at least 95% accuracy(combined hits and correct rejections) must be omitted. Because subjectsare confined to a relatively narrow range of behavior, we refer to thisphase of the process as a “behavioral clamp.”

[0010] The VMT provides fairly good control of subject's overt behaviorand even of their inner behavior (thinking). However, it does nothing tocontrol mood, another variable that could be different under referenceversus external stimuli (e.g. drug treatment) conditions, but as withsleep in the behavioral domain, it would be incorrect to attributemetabolic changes to a drug. In order to minimize the contribution ofmood to metabolic changes that we would observe, we introduced into thePET experiments a standard test procedure. We administer the Profile ofMood States—POMS (McNair et al., 1971), a brief self-administeredadjective check list that has been shown to be sensitive to drug effects(de Wit et al., 1985; de Wit et al., 1986) and other external stimuli.POMS scale scores are determined before and after the placebo and beforeand after the administration of the stimuli treatment. The differencebetween these scores indicates how much mood changed as a result of theadministration of the stimuli or treatment, as opposed to changes due tofatigue, boredom, etc. We recognize that mood is difficult to control,but by measuring it we can incorporate significant mood changes into ourinterpretation of metabolic changes. Where practical, this ofteninvolves separating subjects who change in mood from those who do not(or who change in the opposite direction) and creating different imagesof metabolic change for each group.

[0011] Having deliberately chosen to deal with relatively small signalsdue to our external stimuli or treatment, we were next faced with theproblem of detecting those signals. The standard method of dealing withmetabolic images in PET studies (prior to OMEI) consists of drawinganatomical regions of interest (ROIs) on the slices that the scannerprovides; this is done under both reference and treatment conditions. Inits more recent form (Gut et al., 1995a), the ROIs are drawn on eachsubject's MRI then applied to the PET images that are spatiallycorrelated (in three dimensions) with the MRI. In either form, thismethod is relatively insensitive to small changes in brain metabolism(Fox, 1991).

[0012] 1. Even with the best positioning techniques, there will beslight differences in positioning of subjects on different occasions. Inthe case of repeated 0-15 scans we have even noticed significant changesin subject positions (up to 5 mm) within the same session; this problem,of course, is exacerbated when metabolic studies occur in differentsessions on different days. This means that slices of the brain in onecondition will not correspond exactly to ROIs from another condition.

[0013] 2. The problem of different slices is even more serious whenlooking at different subjects since anatomical differences will preventdefinition of identical ROIs.

[0014] 3. Even the best drawing of ROIs cannot perfectly define allregions identically in all subjects. Not only will experimenter errorand biases be present, but differences in anatomical features will causesome variation in definition of ROIs.

[0015] 4. Any ROI must necessarily include relatively unresponsivesubregions (e.g., white matter, portions near boundaries of ventriclesor external surfaces that incorporate different partial volume effects).

[0016] 5. True physiological effects will often not fill an entire ROI,no matter how small the ROIs may be.

[0017] 6. Because the ROIs are defined independently of each other,physiological effects that cross ROI boundaries may fail to show up inany one ROI even though an effect may be relatively large.

[0018] The first five of these considerations serve to add “noise” tothe signal that we would be trying to detect the sixth effectivelyreduces the size of a signal even further. Nevertheless, to ourknowledge all PET metabolic studies to date, including our own (de Witet al., 1988; de Wit et al., 1991), have used this basic approach. Thisapproach, as we and others demonstrated can work in the sense ofdemonstrating robust effects. When combined with the behavioral clampprocedure, this approach can certainly provide interpretable metabolicimages. At worst, it would only require the studying of a sufficientlylarge number of subjects to determine effects of any external stimuli(e.g. drug) treatment (this, of course, can be practically impossible,given the cost of PET studies).

[0019] Coincidentally with these metabolic studies, however, others wereconducting studies of cerebral blood flow with 0-15. In these studies itwas early recognized that small signals were involved and therefore moresensitive analytic approaches were required. Such approaches weredeveloped in several laboratories (Fox et al., 1988; Fox and Mintun,1989). Underlying these more sensitive methods were two conceptualshifts from the standard procedure.

[0020] The first as a recognition that PET was providing truephysiological data, not anatomical data (Fox, 1991). It stands toreason, therefore, that the physiological data itself would be moresensitive than a priori anatomical features. In this sense, thesemethods were “data driven”. The second shift was an effectiverealization that the slice-based data provided by PET scanners areestimates of true metabolic activity in the brain. Alternative ways ofestimating metabolic activity can be validly employed. Briefly, the PETbrain could be conceived of as a whole, relatively smooth volume ratherthan a set of discrete slices. Of course there are assumptions andlimitations in this volumetric approach, but they are not necessarilyworse than those of the slice-based approach. Most important, thevolumetric approach allows for different kinds of data manipulation inexperimental settings.

[0021] Specifically, estimates can be made for the metabolic value atevery point in the brain and the brain can all be transformed into astandard three-dimensional space. Several different methods have beendeveloped for estimating all points in the brain volume. Basically, theyall involve interpolation from measured slice centers to every point inthe vicinity of the slice center. At present, only linear interpolationshave been employed but other methods are being researched (Lin et al.,1988; Lin et al., 1989). Likewise, the transformation problem has beensolved several times (Evans et al., 1987; Fox et al., 1988; Evans etal., 1991). Investigators in our laboratory have been most successful indeveloping the procedure for spatially correlating PET and MRI or x-rayCT images (Pelizzari et al., 1989). While not essential, this step isone more way of reducing noise due to imprecision of the spacialtransformations. Although we have preferred ways of treating our data,we recognize that there are a number of comparably good techniques. Thecritical point for the OMEI procedure is that volumetric handling of thedata with transformation into standard space is an essential part of theprocedure.

[0022] The present invention involves brain-behavior relationships andmethods for evaluating and measuring them using imaging techniques withpositron emission tomography (PET). In particular we are concerned withmethods that measure quantitative changes in blood flow, metabolism andligand localization and binding. More specifically we have been involvedwith elucidating the effects of external stimuli such as drugs and otherpsychoactive compounds with abuse potential as well as environmentalinfluences like fragrances, temperature, vibration, taste, noise, lightand other sensory-perceived influences, and cognitive challengesconcerning attention and memory. We have effected a method which enablesus to measure regional metabolic changes in the brain and associatedmood changes as a result of administering such external stimuli,particularly from a single-dose drug challenge, in a controlledbehavioral state.

[0023] The method presents a new perspective inasmuch as it reveals theend-pathway of the external stimuli's effect by the metabolic processinvolved. Thus, it demonstrates those regional brain areas which effectthe functional changes induced by the external stimuli, particularly bythe action of a drug or psychoactive compound. Further, it cancharacterize the metabolic changes in quantitative terms as to whetherthe regional metabolic change is relatively increased or decreased. This“end-effect” measure is particularly important since we have shown it isquite distinct from the site of localization of the radiolabeled drug,its ligand-binding characteristics or the neurotransmitter systemsinvolved.

[0024] A series of studies indicate this is an effective in vivo meansof characterizing the effects of external stimuli, particularly drugs,and thus can provide a new and valuable approach to drug development.Specifically, we see an application in devising effective and efficientstrategies in the clinical phases of development; not least from theability to rapidly obtain a measure of effectiveness by directcomparison with already characterized and available compounds. Webelieve the method can be advantageously applied in all three clinicalphases of drug development—safety, efficacy and dosage. It can also be ameans to determine the effects of external stimuli, such as drugcombinations and examine synergy or inhibition. Our special interest isin psychoactive compounds that effect mood and behavior and theirassociated neuropsychiatric disorders, but as noted above the method isapplicable to other types of external stimuli. However, the approach isalso applicable in drugs targeted to broader range of syndromes anddisorders in the brain.

[0025] Similarly, we recognize the potential in determining drugside-effects including CNS changes arising from non-brain targetedpharmaceuticals.

[0026] In terms of drug development, the efficiency of the method is anoutstanding attribute. Significant measures may be obtained from as fewas eight subjects and comparative results provided with a matter ofweeks. It is amenable to many variations in the drug testing procedureincluding measures of acute and chronic effects and alterations indosage, scheduling and delivery. It will provide an important measure ofdrug effect since for the first time it will be possible to relatedosage and drug plasma levels to a quantitative measure of regionalchanges in the central nervous system. Similarly, when these measuresare related to parenchymal organ function of blood biochemistry, thentoxicity versus therapeutic efficacy can be quantitatively appraised.

[0027] These considerations indicate that the method can have asignificant impact upon both the cost and rate at which new drugs can bedeveloped and brought to market.

[0028] Archived compounds can be efficiently re-evaluated and, perhapsmost importantly, it will increase the number and type of new compoundsthat can be applied in the therapy of diseases affecting the brain.

[0029] The method involves (1) measuring cerebral metabolism of asubject's brain prior to and in the absence of any external treatmentwith a stimuli such as a psychoactive compound; (2) administering theexternal stimuli such as a psychoactive compound to the subject; (3)controlling behavioral influences on the subject's brain by subjectingthe subject to a behavioral clamp; (4) measuring cerebral metabolism ofthe subject's brain after administering the external stimuli (e.g.psychoactive compound) and during the behavioral clamp; and (5)determining any differences between cerebral metabolism prior to and inthe absence of administering the external stimuli (e.g. psychoactivecompound) and cerebral metabolism after administering the externalstimuli (e.g. psychoactive compound).

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

[0030] In the drawings:

[0031]FIG. 1 illustrates a series of PET images superimposed on MRIbrain images demonstrating fluoxetine effects on cerebral glucosemetabolism;

[0032]FIG. 2 illustrates a series of PET images superimposed on MRIbrain images demonstrating pimizide effects on cerebral glucosemetabolism; and

[0033]FIG. 3 illustrates a series of PET images superimposed on MRIbrain images demonstrating ethanol effects on cerebral glucosemetabolism.

DETAILED DESCRIPTION OF THE INVENTION

[0034] The Optimization of Metabolic Effect Identification (OMEI)procedure disclosed herein uses positron emission tomograph (PET) forthe purpose of identifying regions of the brain that are effected by anexternal stimuli or treatment. The external stimuli or treatment willusually be a pharmacological treatment but the procedure is not limitedto this type of study and may be employed to determine the effects ofany psychoactive compound on a patient's brain. For example, pimizide,ethanol, fluoxetine, perfumes, taste or flavoring compounds, etc. areall examples of psychoactive compounds. Other environmental influencessuch as temperature, vibration, noise, light and other sensory-perceivedinfluences may also be evaluated for their effects on cerebralmetabolism. The procedure is effective because it reduces the influenceon metabolism of effects not directly due to the treatment being studiedand because it uses the most sensitive available methods for detectionof areas that change in metabolism because of the treatment. Thus, thephrase “external stimuli or treatment” is intended to be anypharmaceutical drug, psychoactive compound or sensory-perceivedenvironmental influence.

[0035] The procedure consists of four steps:

[0036] 1. Collection of Metabolic Data: Two or more PET sessions areconducted for every subject. In each session, fluoro-deoxyglucose (FDG)is used as the tracer to measure regional cerebral metabolism of glucoseaccording to standard methods (Phelps et al., 1979; Reivich et al.,1979). One of these sessions is designated as the reference session. Inmost cases, the reference session includes the administration of aninert treatment (placebo); the other session(s) include theadministration of an external stimuli or treatment such as a drug (ordrugs) which is assumed or hypothesized to affect the brain. Ideally,the treatments should be administered with a placebo in a double-blind,counterbalanced fashion at a time prior to administration of the dose ofthe external stimuli or treatment (within limits—see below) whichpermits determination of time-activity or dose-response curves. PET datacan be collected in any convenient units (e.g. absolute units of glucoseper unit tissue per unit time, or normalized relative to whole brainmean and standard deviation), although the ultimate interpretation willbe affected by the choice of units.

[0037] 2. Control of Behavioral and Subjective Influences on the Brain:In each PET session the subject performs a behavioral task which has oneor more measurable variables. The behavioral task is chosen so as tomaintain subjects in a standard and stable condition throughout theperiod of FDG equilibration (20-40 Minutes after injection). The taskmust be capable of being performed at comparable levels by all subjectswith or without the external stimuli or treatments that may be used inthe study. Thus, the task should be relatively simple, requiring anon-stressful level of attention with no opportunity for developing aproductive strategy. The measurable variables associated with the taskare used to define criteria for a valid study: only subjects who performthe task at a specified level are included in subsequent analyses.Because subjects are behaviorally restricted to a relatively narrowrange of responses, we refer to this step as a “behavioral clamp”.Implicit to this approach is a recognition that only certain levels ofthe treatment can be studied by this method. For example, a dose of adrug which invariable produces sleep or uncontrollable agitation couldnot be studied. In our usual application, the clamp consists of visualmonitoring task (VMT) is which the subject is asked to press a buttonheld in one hand every time a dim light is flashed on a screen but toignore an equally probably and easily discriminated bright flash. Brightor dim lights flash randomly for 250 msec at varying intervals between 4and 7 seconds. Arbitrarily, we set the validity level at 95% accuracywith reaction times varying in experimental conditions by no more than±10% from the reference condition for each subject.

[0038] The task should also minimize subjective variables such asextraneous ideation, motivation, anxiety, etc. In part, this isaccomplished by the choice of a behavioral clamp which is sufficientlybut not excessively demanding; this is verified by the overall stabilityof performance by subjects across a wide range of treatments andsubjects. In addition, some subjective variables can be quantified bystandard psychological instruments. We administer the Profile of MoodStates (POMS) (McNair et al., 1971) before and immediately after theequilibration period. If there is a significant group difference as afunction of treatment, the subjects are divided into subgroups so as tofacilitate attribution of image differences to the measured subjectivedifferences. Thus, there are two components to this step: clamping thebehavior and measuring mood.

[0039] 3. Transposition of Image Data into Standard Computer Space:Metabolic image data collected from each subject are transposed into astandard anatomical space. This involves shifting, shrinking orexpanding, and rotating PET image sets so that any point within theimage-brain of one subject corresponds (in x, y, and z coordinates) tothe same point in the image-brain of that subject under differentconditions and to the same point in the image-brains of all othersubjects. Several methods could be used to achieve this. We use a set ofstandard anatomical landmarks which can be identified in each threedimensional image-brain. The landmarks are then adjusted to correspondto their location in the Talairach atlas (Talairach and Tournox, 1988).This process is facilitated by the collection of anatomical image-brainwith the metabolic image-brain in computer space. We use the method ofPelazzari and colleagues, which was developed in our laboratory(Pelizzari et al., 1989); other methods could also be used. Thelandmarks are then identified in the anatomical image-brain, applied tothe metabolic image-brain, and transformed into the Talairach space.

[0040] 4. Detection of Areas of Treatment Effects: A number of methodscan be used to distinguish areas of the brain that show markeddifferences between the treatment conditions. We use a modification ofstatistical parametric mapping (Friston et al., 1991) in whichwithin-subject t-tests are performed at each voxel in the standardizedimage-brains for each stimulated condition compared to the referencecondition. The level for reporting differences is set depending on therequirements of the study. Simpler analyses, such as subtractions of thetreatment mean images from the reference mean image can be used forpreliminary analyses.

[0041] We claim a unique contribution especially for the development ofstep 2. The other steps are widely employed and accepted in the PETliterature. We also claim the first recognition of the usefulness of thesteps in combination as applied to the problem of evaluating the effectsof an external stimuli or treatment on cerebral metabolism.

[0042] The result of this procedure is to improve the ability to detectchanges in a subject's neuronal energy consumption that can beattributed to an external stimuli or treatment. The term “'subject” isintended to include patients, normal volunteers, and also non-hurnanmammals such as primates. The procedure does this by reducing theinfluence on metabolism of extraneous factors (i.e., those factors notspecific to the treatment or secondary to the treatment) which wouldotherwise provide metabolic changes greater than those attributable tothe treatment itself. Step 2 (Behavioral control), specifically,minimizes behavioral and subjective influences on metabolism. Step 3 wasoriginally developed for use in PET studies of cerebral blood flow underconditions of sensory or cognitive activation. The merits of thisapproach in detecting physiological changes compared to the anatomicalbased methods which are the alternative “state of the art” have beendiscussed in the literature (Fox, 1991).

[0043] Accordingly, the present invention provides a method ofevaluating the effects on a subject's brain of administering an externaltreatment to a subject comprising the following steps:

[0044] 1. Measuring cerebral metabolism of a subject's brain in theabsence of any external treatment administration;

[0045] 2. Administering an external treatment to the subject;

[0046] 3. Controlling behavioral influences on the subject's brain byusing a behavioral clamp, said behavioral clamp comprising a procedurethat:

[0047] (a) Maintains the subject to a standard and stable conditionthroughout a period of accumulation of cerebral metabolic informationwith and without said external treatment;

[0048] (b) Is capable of being performed by the subject at comparablelevels prior to as well as subsequent to administration of said externaltreatment;

[0049] (c) Requires a non-stressful level of attention with noopportunity for the subject to develop a productive strategy;

[0050] (d) Includes measurable variables capable of defining desiredcriteria; and

[0051] (e) Minimizes subjective variables including extraneous ideation,motivation, and anxiety.

[0052] 4. Measuring cerebral metabolism of the subject's brain afteradministering the external treatment and during the behavioral clamp;and

[0053] 5. Determining any differences between cerebral metabolism in theabsence of administering the external treatment and cerebral metabolismafter administering the external treatment.

[0054] The location of metabolic changes which we have noted with thisprocedure do not necessarily correspond to known locations of receptorsfor the drugs that we have tested. Instead, they correspond to regionsof maximal metabolic change due either to direct or indirect effects ofthe treatment.

EXAMPLE 1

[0055] This study was conducted to determine the effects of oraladministration of 40 mg of fluoxetine, an agent which inhibits re-uptakeof 5-HT, on CMRglu, as measured by positron emission tomography (PET)using (¹⁸F)-2-deoxyglucose (FDG) in healthy human subjects.

[0056] Materials and Methods

[0057] Subjects: Four healthy control subjects were studied. Fivepotential subjects were screened for personal psychiatric history usingthe SCID-NP⁵ and a clinical mental status examination. They werescreened for mental illness with a history and physical examination.Potential subjects were excluded if they had Axis I disorders, substanceabuse, alcohol abuse, or significant medical illness, and one wasexcluded because of a previous episode of major depression. None of thesubjects who underwent the fill procedures had taken psychotropicmedication. Three of the subjects were male and one was female. The ageof the subjects range from 20 to 39 years.

[0058] Procedures:

[0059] All PET studies were performed at the Franklin McLean MemorialResearch Institute of the University of Chicago using a 3-ring PETT VIscanner. Each subject participated in two PET sessions. Subjectsabstained from beverages containing sugar or caffeine and food for atleast 4 hours preceding each study. Fluoxetine (40 mg) or matchedplacebo was administered orally in a double-blind, counter-balancedmanner between 11.00 h and 13.00 h. At each session, the subject waspositioned in the PETT VI in such a way that slices parallel to theorbital-meatal plane were obtained. A laser beam apparatus andcustom-made plastic face mask assured precise and reproduciblepositioning. A transmission scan was performed in each session forattenuation correction. Intravenous catheters were inserted in each armfor blood sampling and for injection of the radioactive tracer.

[0060] Ninety minutes after administration of fluoxetine or placebo,subjects were repositioned in the PET scanner and a visual monitoringtask (VMT) was initiated. The task assured a stable behavioral conditionat the time of testing. Each subject was asked to press a button withhis or her right thumb every time a dim light (50% of trials) waspresented on a screen mounted in front of him or her and was asked toignore every bright light (50%). The lights were presented undercomputer control at random intervals ranging from 4 to 7 sec. Eachsubjected completed four blocks of 96 trials each (approximately 10 minper block). Subjects also completed the Profile of Mood States (POMS)before the capsule and 20 min after injection of FDG.

[0061] FDG (6.0-7.5 mCi) was administered 30 sec after the initiation ofthe visual monitoring task and static scanning commenced 40 min laterfor a period of 14 min. Five simultaneous planes were obtained with aninterplane separation of 14 mm. Subjects were then repositioned bymoving the subject chair of acquisition of an additional five slices instatic mode to more full sample the whole brain. In-plane resolution ofPETT VI is 8 mm at full width maximum.

[0062] Data Analysis:

[0063] PET images were reconstructed using standard methods. Slices fromthe two scanning positions were combined into a single volume for eachsubject. Average CMRglu was estimated as the mean of all slices,excluding voxels having 60% or less of the maximum metabolic rate(assumed to be ventricles, white matter, and non-brain tissue). Averageglobal CMRglu was compared between placebo and fluoxetine scans usingthe paired two-tailed t-test.

[0064] All voxels were normalized and expressed as a z-score valuerelative to the whole scan mean and standard deviation of gray mattervoxels. PET volumes for each subject were spatially correlated acrossthe two conditions using the surface-fitting technique developed in ourlaboratory.

[0065] PET volumes were also anatomically normalized, i.e., eachsubject's images were expanded, contracted, rotated, or shifted into astandard volume using the coordinate system of the Talairach atlas, wefirst identified a set of landmarks which were used to proportionallyadjust the entire PET volume into “Talairach space”. In Talairach space,the PET slices were linearly interplated between each measured slice toprovide better localization of anatomical features.

[0066] In the Talairach volumes, two-tailed paired t-tests wereperformed comparing each voxel from placebo condition to each voxelafter fluoxetine. Paired t-tests were used because each subject wastested in each condition. A t-value of 3.2 closely approximatedstatistical significance for 3 degrees of freedom. To adjust for thelarge number of voxels studied, we only considered regions significantif a large number of adjacent voxels had t-values greater than 3.2 orless than −3.2. Determination of localization of areas of increased ordecreased metabolism were performed by displaying significant voxels incolor overlaid on a gray scale MRI.

[0067] Results:

[0068] Average global CMRglu was not significantly different betweenplacebo (8.93±0.96 mg 100 g¹ min⁻¹) and fluoxetine scans (8.22±0.86 mg100 g¹ min⁻¹⁻, paired t=0.82, df3, p<0.48). Inspection of the t-testimages revealed that two areas had marked changes in relative glucosemetabolism. Most prominent was a bilateral C-shaped region consisting ofamygdaloid complex, hippocampal formation, and ventral striatum whichshowed decreased relative glucose metabolism. A smaller area centered inthe right superior parietal lobe (Brodmann area 7) showed increasedrelative metabolism (FIG. 1).

[0069] There were no systematic effects of fluoxetine on reaction timeor accuracy on the VMT (median reaction time 571±s.d. 125 ms afterplacebo, 553±64 msec after fluoxetine, t=−0.78; 91.7±14.6% correct afterplacebo, 96.4±3.2% correct after fluoxetine, t=0.82). There were nodifferences in subjective effects as measured by the POMS.

[0070] Discussion:

[0071]FIG. 1 illustrates statistical maps of voxel-by-voxel repeatedmeasures t-tests for the data obtained after subjects had receivedfluoxetine and placebo. PET images are superimposed on a normal MRIimage in Talairach coordinate space, thresholded to show only (valuesgreater than 3.2 (red) or less then −3.2 (blue X)(p<0.05). Red areasshow regions in which the fluoxetine condition had greater relativemetabolism than placebo; blue areas are regions in which the fluoxetinecondition had less relative metabolism than placebo. In each axialslice, the front of the brain is at the top, the left side of the brainis to the left. Each successive slice (from left to right, then top tobottom) shows progressively lower slices in brain, beginning atTalairach coordinates approximately 3.4 cm above AC-PC line; slices areapproximately 4.0 mm apart. Sagittal slices begin at the far left of thebrain and proceed from left to right, top to bottom in approximately 6.5mm steps through the midsagittal plane (middle slice in second row) tothe far right side. Administration of fluoxetine led to a decrease inCMRglu in bilateral amydeloid complex, hippocampal formation and ventralstriatum and an increase in metabolism centered in the right superiorparietal lobe (Brodmann area 7).

EXAMPLE 2

[0072] In this study we investigated the brain's response to thepsychoactive compound pimizide. Subjects were tested under behavioralconditions substantially identical to the procedures described inExample 1, except pimizide was administered instead of fluoxetine.

[0073]FIG. 2 shows changes in rCMglu demonstrating the effects ofpimizide

EXAMPLE 3

[0074] In the present PET study we tested subjects to investigateindividual differences in response to ethanol. Subjects were tested withplacebo (tonic water) and with a moderate dose (0.5 g/kg) of ethanolunder comparable behavioral conditions substantially in accordance withthe procedures described in Example 1.

[0075]FIG. 3 shows changes in rCMglu demonstrating the ethanol effects.The most striking effect was a widespread increase in rCMglu in the lefthemisphere. Other areas affected were in the frontal and temporal lobes,basal ganglia, and limbic system.

EXAMPLE 4

[0076] In this study we investigated the brain's response to thepsychoactive compound nimodopine. Nimodopine (0.5 g/kg) or placebo (mixalone) was administered in a 250 ml beverage in lime juice and tonicwater to be consumed in five minutes. All other behavioral conditionswere substantially identical to the procedures described in Example 1.

[0077] The data appear to show decreased metabolism in the cingulategyrus and/or left posterior temporal lobe with patchy increasedmetabolism in the superior portion of the cerebellum and/or interiorportion of the occipital lobe.

I claim:
 1. A method of evaluating the effects of administering anexternal stimuli to a subject on the subject's brain, comprising thesteps of: measuring cerebral metabolism of a subject's brain prior toany treatment with an external stimuli; administering the externalstimuli to the subject; controlling behavioral influences on thesubject's brain by subjecting the subject to a behavioral clamp;measuring cerebral metabolism of the subject's brain after administeringthe external stimuli and during the behavioral clamp; and determiningany differences between cerebral metabolism prior to administering theexternal stimuli and cerebral metabolism after administering theexternal stimuli.
 2. The method of claim 1 wherein said external stimuliis a psychoactive compound.
 3. The method of claim 2 wherein thecompound administered is a fragrance.
 4. The method of claim 2 whereinthe compound administered is fluoxetine.
 5. The method of claim 2wherein the compound administered is ethanol.
 6. The method of claim 2wherein the compound administered is pimizide.
 7. The method of claim 2wherein the compound administered is nimodopine.
 8. The method of claim1 wherein the step of measuring cerebral metabolism prior to anytreatment utilizes position emission tomography.
 9. The method of claim1 wherein the step of measuring cerebral metabolism after administeringthe external stimuli utilizes position emission tomography.
 10. Themethod of claim 1 wherein said behavioral clamp comprises a visualmonitoring task performed by the subject.
 11. The method of claim 1further including the step of measuring a subject's mood both prior toand after administering the external stimuli.
 12. The method of claim 1wherein the step of measuring a subject's mood comprises administering astandard psychological test thereto.
 13. The method of claim 12 whereinsaid standard psychological test is a profile of mood states.
 14. Themethod of claim 1 further including the step of comparing the cerebralmetabolism after administering the external stimuli to a referencecerebral metabolism.
 15. The method of claim 1 wherein said externalstimuli is a pharmaceutical drug.
 16. The method of claim 1 wherein saidexternal stimuli is an environmental influence.
 17. The method of claim16 wherein said environmental influence is selected from the groupconsisting of temperature, vibration, noise, and light.
 18. A method ofevaluating the effects on a subject's brain of administering an externaltreatment to a subject comprising the following steps: measuringcerebral metabolism of a subject's brain in the absence of any externaltreatment administration; administering an external treatment to thesubject; controlling behavioral influences on the subject's brain byusing a behavioral clamp, said behavioral clamp comprising a procedurethat: (a) maintains the subject in a standard and stable conditionthroughout a period of accumulation of cerebral metabolic informationwith and without said external treatment; (b) is capable of beingperformed by the subject at comparable levels prior to as well assubsequent to administration of said external treatment; (c) requires anon-stressful level of attention with no opportunity for the subject todevelop a productive strategy; (d) includes measurable variables capableof defining desired criteria; and (e) minimizes subjective variablesincluding extraneous ideation, motivation, and anxiety; measuringcerebral metabolism to the subject's brain after administering theexternal treatment and during the behavioral clamp; and determining anydifferences between cerebral metabolism in the absence of administeringthe external treatment and cerebral metabolism after administering theexternal treatment.
 19. The method of claim 18 wherein said externaltreatment is selected from the group consisting of a psychoactivecompound, a pharmaceutical drug and an environmental influence.